PROJECT SUMMARY Diffuse gliomas comprise the majority of malignant primary brain tumors and are commonly fatal. In a quest to better understand their biology, many glioma-associated mutations have been identified over the past decade. Among these, we have shown that germline mutations in POT1 are associated with increased risk of glioma development. POT1 is known to bind and protect telomeres, and several POT1 mutations in other cancers are known to result in telomere elongation. However, it is not known whether the glioma-associated POT1 mutations can cause telomere elongation, and more fundamentally, the mechanisms linking germline POT1 mutations to glioma initiation are not understood. The overall goal of this proposal is to determine how POT1 mutations facilitate glioma initiation. I hypothesize that POT1 mutations accomplish this by (1) driving cell proliferation and (2) enabling telomere elongation and bypass of replicative senescence. We plan to use glial progenitor cells (GPCs) as a model for glioma initiating cells. We will use mouse GPCs to address the first hypothesis, as mice allow in vivo modeling, and human GPCs to address the second hypothesis, as human cells are more readily capable of replicative senescence. My first Specific Aim seeks to quantify the effects of POT1 mutant expression or loss of mouse POT1 orthologs in GPC proliferation and fate in vivo, using an in utero electroporation mouse model, as well as in vitro, at various time points into adulthood. We will also assess these effects in lineage-restricted GPCs using selective promoters to drive gene expression. My second Specific Aim seeks to establish the role of POT1 mutations in replicative senescence bypass using human GPCs. We will assess the effects of POT1 mutant expression or POT1 loss on telomere length and population doubling capacity of human GPCs. We will also quantify the tumorigenesis potential of these cells in a xenograft model upon inhibition of p53 and activation of Ras pathways. These studies will help to reveal the role of POT1 mutations in glioma initiation. In the long term, I intend to leverage this knowledge to gain further insight into the mechanisms of telomere biology in glioma and translate its vulnerabilities into potential therapeutic measures. I envision a career for myself as an independent physician-scientist studying the biology and genetics of gliomas with an active brain tumor surgical practice focused on treating patients with glioma. To reach this goal, I have identified several areas of deficiency in my training that require addressing. These include knowledge of telomere biology and genetic engineering techniques as well as bench and laboratory management skills. I plan to address these deficiencies by learning from my mentor and colleagues as well as participation in courses and conferences. Baylor College of Medicine and Department of Neurosurgery provide a highly fertile environment for research, and I enjoy the full support of my mentor, advisors, and Department Chair. The program outlined in this proposal will provide me with the training to successfully complete my proposed studies as well as the track record to compete for research funding as an independent investigator.